Project Summary It is widely believed that damaged axons in the adult mammalian brain have little capacity to regrow, thereby impeding functional recovery after injury. Serotonin neurons appear to be a notable exception. We have produced time-lapse images of serotonin axons in the neocortex of the adult mouse. Serotonin axons undergo massive retrograde degeneration following amphetamine treatment and show subsequent slow, long-distance regrowth. A stab injury that transects serotonin axons running in the neocortex is followed by local regression of cut serotonin axons and by regrowth from cut ends that projects across the stab rift zone. By contrast, dopamine fibers originating in neurons of the ventral tegmental area (VTA) are also small-diameter unmyelinated axons, yet they are not damaged by amphetamine treatment and they fail to regrow after stab transection. What are the molecular specializations that allow serotonin axons to regrow while other injured axons in the brain fail to do so? To begin to address this question we have harvested pools of serotonin neurons from the dorsal raphe of adult mice in order to measure gene expression using genome-wide RNA sequencing (RNA-Seq). This was done 1 week after the end of either amphetamine or saline treatment. Our preliminary data have revealed a set of genes that are significantly upregulated in serotonin neurons during amphetamine-evoked axon regrowth. Here, we propose to complete and extend this screen as a first step to define the molecular requirements for serotonin axon regrowth. Aim 1. What genes are differentially expressed in serotonin neurons during amphetamine-evoked regrowth of their axons in the neocortex? Our preliminary data reveal ~50 upregulated and ~60 downregulated genes measured ~1 week after the end of amphetamine treatment as compared with saline-treated-controls (using a false-discovery rate of p<.05). To complete this data set, we shall extend these RNA-Seq measurements using a time course of amphetamine and saline-treated mice at t= 1 day, 1 week and 18 weeks. There will be 8 animals per group (4 male, 4 female), and we shall validate genes of interest with in situ hybridization and immunohistochemistry. We shall also compare the gene expression changes in response to amphetamine challenge in serotonin neurons with those changes produced in dopamine neurons of the VTA. Aim 2. What genes are differentially expressed in serotonin neurons during regrowth of serotonin axons following a neocortical stab lesion? In contrast to amphetamine lesions, stab lesions produce only local axonal regression of serotonin and dopamine axons. Stab lesions form a glial scar, as occurs in stroke and other forms of acute brain injury. We shall compare dorsal raphe serotonin neuron and VTA dopamine neuron expression profiles in response to stab and amphetamine injury using the same post-injury time points. The results of these two complementary injuries in regenerating and non-regenerating neurons will inform later manipulative experiments to modulate serotonin axon regrowth and, ultimately, confer the capacity to regrow on non-serotonergic neurons in the brain.